Casein peptide for use in the treatment of uterine infections

ABSTRACT

The present disclosure relates to a casein peptide for use in the treatment of an uterine infection in a female mammalian animal, to methods of treatment of such infections by administering to a female mammalian animal at least one casein peptide, to the use of casein peptide for the preparation of a pharmaceutical composition for treatment of uterine infection and to a kit for said treatment. The casein peptide is preferably a casein hydrolysate, e.g. obtained by trypsin hydrolysis of casein protein. The female mammalian animal is, in accordance with some embodiments, a lactating animal, the infection being a post-partum infection.

FIELD OF THE INVENTION

The present invention relates to casein peptides and to therapeutic usesthereof.

BACKGROUND OF THE INVENTION

The casein protein comprises three fractions, α, β and γ, according totheir electrophoretic mobility. Casein hydrolysate is the hydrolyzedform of casein which includes, among others, the activebeta-casein-derived peptide. It has been established that caseinhydrolysate plays a role in immune responses against microbial and viralinfections.

For example, U.S. Pat. No. 3,764,670 describes the anti-bacterialactivity of casein fragments (obtained by proteolytic activity) anddiscloses proteolytic casein digests possessing antimicrobial propertiesagainst microorganisms.

U.S. Pat. Nos. 5,506,209, 5,538,952 and 5,707,968, all to Mukerji etal., and 5,968,901 to Andersson, et al., describe the administration ofonly human beta-casein, recombinant human beta-casein, and hydrolysatesof both, in a liquid enteral formula, for treating respiratory syncytialvirus, otitis media, H. influenza and other infections in infants.

U.S. Pat. No. 5,330,975 describes the use of sialic-acid bindingkappa-casein and kappa-casein peptides for the neutralization ofbacterial endotoxins, such as cholera toxin.

U.S. Patent Application publication No. 2009/0305947 describe the use ofcasein peptides and in particular casein hydrolysate for the managementof the reproductive cycle of livestock and farm animals. U.S. PatentApplication publication No. 2009/0069218 describe the use of caseinpeptides and in particular casein hydrolysate as a method for decreasingthe length of the dry period of a lactating livestock animal, forincreasing its milk yield and milk hygiene after parturition and forimproving the livestock welfare.

SUMMARY OF THE INVENTION

In accordance with a first of its aspects, the present disclosureprovides a casein peptide for use in the treatment of a uterineinfection in a female mammalian animal. Such treatment facilitates,inter alia, good postpartum management in mammalian animals

The present disclosure also provides, in accordance with a second of itsaspects, a method for treatment of a uterine infection, the methodcomprises administrating to a female mammalian animal in need oftreatment of uterine infection, an amount of at least one caseinpeptide, the amount being effective to treat an uterine infection in themammalian animal

The present disclosure also provides, in accordance with a third of itsaspects, the use of at least one casein peptide, for the preparation ofa pharmaceutical composition for the treatment of a uterine infection ina female mammalian animal in need of treatment of uterine infection.

In addition, the present disclosure also provides, in accordance with anaddition aspect a pharmaceutical composition for the treatment of auterine infection, the composition comprising as an active ingredient anamount of at least one casein peptide, the amount being effective totreat uterine infection in a female mammalian animal.

Finally, in accordance with an additional aspect, the present disclosureprovides a kit comprising at least one casein peptide and instructionsfor use the casein peptide for the treatment of a uterine infection in afemale mammalian animal

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based on the finding that casein peptides, andin particular, casein hydrolysate can be used for the treatment ofuterine infections.

Specifically it was found that administration of casein hydrolysate topostpartum cows was effective in the treatment of clinical metritis.

The uterus of mammalian animals, such as bovine, is sterile prior tocalving. During gestation, the cervix provides a barrier againstintra-uterine invasion by pathogens. At calving, a wide range ofbacteria enter the uterus via the now open cervix offsetting a cascadeof events that culminate in uterine infection, inflammation and,finally, uterine clearance. However, pathogenic bacteria persist in somecows causing uterine disease by preventing physiological uterineinvolution resulting in sub-fertility. Because reproductive performanceis critical for viable dairy production and reproduction of mammals,uterine health in the post-partum period requires substantial medicalveterinary attention.

Parturition-conception intervals and thus the pregnancy rates are acritical factor from an economical point of view of livestock and farmmanagement. The ability to maintain an optimum period ofparturition-conception intervals of about 12 to 14 month depends onnumerous factors including, inter alia, the clinical state of theanimal's uterus, which should be completely free of any post-partumrelated infections.

Metritis, endometritis, and pyometra are common uterus inflammatorydiseases, that occur during the postpartum period of livestock and farmanimals, mainly in the early postpartum period, and are primarilyassociated with contamination of the reproductive tract i.e. uterine.

Uterine infections, and retarded uterine and cervical involution causeseconomic losses due to costs for treatment, milk withdrawal, reducedreproductive performance, and premature culling.

A wide variety of therapies for endometritis have been reported,including mainly antibiotics administered either by systemic or localadministration of prostaglandin F2α (PGF2α) or estradiol.

The present disclosure provides an alternative treatment for uterineinfection. Thus in accordance with the first aspect, the presentinvention provides a casein peptide for use in the treatment of auterine infection in a female mammalian animal. As appreciated, whilethe invention is described in the following detailed description withreference to the casein peptide for use in the treatment of an uterineinfection in a female mammalian such as a lactating animal, it is to beunderstood that also encompassed within the present disclosuretherapeutic methods comprising administration of the casein peptide tofemale mammal animal, as well as to pharmaceutical compositionscomprising as an active ingredient an amount of at least one caseinpeptide, the amount being effective to treat uterine infection; and tothe use of a casein peptide for the preparation of a pharmaceuticalcomposition for the treatment of uterine infection in said animals.

Casein is the principle protein in non-human mammal's milk, also foundin human mammal's milk known to include the subgroups αS1, αS2, β and κ.Casein is defined according to the amino acid sequences of each of thesubgroups αS1, αS2, and κ (Farrell, Jr. H. M., Jimenez-Flores R., BleckG. T., Brown E. M., Butler J. E., Creamer L. K., Hicks C. L., Hollar C.M., Ng-Kwai-Hang K. F., Swaisgood H. E. Nomenclature of the Proteins ofCows' Milk—Sixth Revision. J. Dairy Sci., 87:1641-1674 (2004)).

Casein suitable for use in the present invention can be derived from anyof a variety of sources, such as, without being limited thereto, theαS1, αS2 and β and κ-casein of any mammal derived milk, including humanand non-human animals.

In the context of the present disclosure, when referring to casein, itis to be understood as also including acid casein, salts of casein,phosphorous containing casein and rennet casein. Further, it is to beunderstood to encompass plant casein analogs. [e.g. as described in U.S.Pat. No. 3,930,058; Kumar, Surinder and Ramachandran, Kolar S. Modifiedvegetable protein simulating casein].

The term “casein peptide” in its broadest sense refers to peptidefragments or peptido-mimetic products obtained from or corresponding toone or more sections of casein protein. The peptide may include, withoutbeing limited thereto, a casein protein breakdown product which occurswhen casein protein is cleaved by enzymes or acids to peptide fragments(also known in the art by the term “casein hydrolysate”) or may refer toa synthetically produced products, where the casein peptide exhibits atleast a therapeutic beneficial effect in treating uterine infection.

The casein peptide may be a single peptide or a mixture of differentpeptides which may be independently naturally occurring, semi-synthetic,synthetic peptide, phosphor-peptide genetically engineered caseinpeptides as well as peptido-mimetics of casein peptides.

The modified, synthetic, semi-synthetic or other types of analogs of thenaturally occurring casein peptides are at least 75%, at times 85%, 90%,95% and even 99% identical (in sequence) to a naturally occurring caseinpeptide when the two sequences are optimally aligned. Further, anynon-naturally occurring casein peptide to be used in accordance with theinvention should retain at least part of the biological activity of thenaturally occurring casein protein.

A casein peptide in accordance with the invention is characterized by amolecular weight of between about 100 to 10,000 Dalton (e.g. between 2to 100 amino acids) and preferably between about 100 to 7,000 Dalton.

In the context of the present disclosure, a casein peptide alsoencompasses a casein hydrolysate. A casein hydrolysate is to beunderstood as the hydrolyzed form of casein (protein), which includes,among others, the active beta-casein-derived peptide known to thoseversed in the art.

Naturally occurring casein peptides are typically obtained followingenzymatic hydrolysis, the enzyme may be any mammal peptidase, such as,without being limited thereto, plasmin, pancreatin, trypsin,chymotrypsin, neutrase, alcalase, pepsine, carboxypeptidase, cathepsinas well as plant peptidase such as, without being limited thereto,papin, bromelain, as well as enzymes from microorganism source. Forexample, a naturally occurring casein peptide may be the result ofplasmin activity on casein subunits β-casein and αs1- and αs2-casein,κ-casein.

In some embodiments, a casein hydrolysate is obtained by cleavage of thecasein protein with tryspin.

The casein peptide may also be a synthetic or semi synthetic peptide. Asynthetic peptide may be obtained by any methods known in the art ofpeptide synthesis, including, solid phase peptide synthesis (R. B.Merrifield (1963). “Solid Phase Peptide Synthesis. I. The Synthesis of aTetrapeptide”. J. Am. Chem. Soc. 85 (14): 2149-2154).

A semi-synthetic casein peptide may be obtained by chemical hydrolysisof casein, e.g. by prolonged boiling in a strong acid (acid-HVP) orstrong base or using a chemical agent such as Cyanogen bromide (CNBr).

The casein peptide may also be obtained by molecular engineering, e.g.using recombinant DNA, in molecular techniques known in the art.

The casein peptide may also include a chemical modification of anaturally occurring peptide, e.g. where one or more amino acids aredeleted, substituted or modified, e.g. by removal of a side group,substitution of a side group or the introduction of a chemical group.Without being limited thereto, the chemical modification may include thefollowing but not exclusive examples: acetylation, acylation, amidation,ADP-ribosylation, glycosylation, GPI anchor formation, covalentattachment of a lipid or lipid derivative, methylation, myristlyation,pegylation, prenylation, phosphorylation, ubiqutination, or any similarprocess. When referring to replacement of an amino acid sequence byanother, it is preferable that the replacement is a conservativesubstitution. For example, one or more amino acid residues within acasein sequence is substituted by another amino acid of a similarpolarity or charge. For example, the non-polar (hydrophobic) amino acidsinclude alanine, leucine, isoleucine, valine, proline, phenylalanine,tryptophan and methionine. The polar neutral amino acids includeglycine, serine, threonine, cysteine, tyrosine, asparagine, andglutamine. The positively charged (basic) amino acids include arginine,lysine and histidine. The negatively charged (acidic) amino acidsinclude aspartic acid and glutamic acid. Nonetheless, non-conservativesubstitutions may also take place as long as it does not significantlychange the desired (casein like) biological activity of the resultingcasein peptide analog.

The casein peptide may also be a peptido-mimietic peptide, such aspeptoids and semipeptoids, which are peptide analogs, having, forexample, modifications such as, but are not limited to, cyclization, Nterminus modification, C terminus modification, peptide bondmodification, including, but not limited to, CH₂—NH, CH₂—S, CH₂—S—O,O—C—NH, CH₂—O, CH₂—CH₂, S—C—NH, CH—CH or CF—CH, backbone modificationand residue modification (methods for preparing peptido-mimeticcompounds may be found in Quantitative Drug Design, C. A. Ramsden Gd.,Chapter 17.2, F. Choplin Pergamon Press (1992), which is incorporated byreference as if fully set forth herein).

The casein peptide according to the invention may comprise “L” as wellas “D” form residues.

In one embodiment, the casein peptide is a single peptide or mixture ofa phosphopeptide, namely, which contains a single phosphorous group oris a phosphorus-enriched peptide. In one embodiment, the casein peptideis any phosphoserine, phosphotyrosine, phosphothreonine, and/orphosphohystidine-enriched casein peptides (casein phosphopeptide, CPP)and monovalent cation phosphocaseinates, such as sodium, potassium,calcium or ammonium phosphocaseinates. In one embodiment, the caseinpeptide enriched with phosphorous groups is a casein hydrolysate In oneembodiment, the casein peptide comprises a motif defined by the aminoacid sequence Ser-Ser-Ser-Glu-Glu (SEQ ID NO:1), where one or more Serresidues are optionally phosphorylated. A non-limiting group of caseinpeptides comprising the phosphorylated motif is provided by SEQ ID Nos:2-6. The following provides SEQ ID NO:2-6, their source and location(residue numbers) in the casein polypeptide:

SEQ ID NO: 2 RELEELNVPGEIVES(p)LS(p)S(p)S(p)EESITR(location 1-25 in β-casein) SEQ ID NO: 3 QMEAESIS(p)S(p)S(p)EEIVPDSVEQK(location 59-79 in αS1-casein) SEQ ID NO: 4KNTMEHVS(p)S(p)S(p)EESIISNETYK (location 1-21 in αS2-casein)SEQ ID NO: 5 KVNELSKNIGS(p)ES(p)TEDQ (location 36-52 in αS1-casein)SEQ ID NO: 6 PTLNREQLS(p)TS(p)EENSKKTVD (location 12-140 in αS2-casein).

While the amino acid residues of the peptide sequences set forth in SEQID NOs:1-6 are all in the “L” isomeric form, residues in the “D”isomeric form can substitute any L-amino acid residue so long as theresulting peptide analog retains at least part of the biologicalactivity of the corresponding “L” isomer. One reason for designingcasein peptides comprising at least one D-amino acid is to increasestability of the peptide to proteolytic degradation.

As indicated above, the casein peptide is used to treat uterineinfection. The term “uterine infection” is used herein to denote apathological condition caused by an influx of pathogens into themammalian animal's uterine and thereby inflammation of the walls of theuterine. In one embodiment, the uterine infection is a pathogen inducedinflammation, also known by the term non-sterile inflammation.

Metritis, either ‘puerperal’ or ‘clinical’; Endometritis, eitherclinical or sub-clinical; and Pyometra are common uterus inflammatoryinfectious diseases that occur mainly during the postpartum period oflivestock and farm animals, mainly in the early postpartum period, andare primarily associated with contamination of the reproductive tracti.e. uterine.

Metritis is an inflammatory reaction involving all layers of the uterus(endometrial mucosa and submucosa, muscularis, and serosa). Animals withmetritis (or perimetritis) have an abnormally enlarged uterus and apurulent uterine discharge detectable in the vagina, more prevalentwithin 21 days post partum, are usually not systemically septicemic,with overt signs of illness (fever, depression, weakness, andin-appetence).

Puerperal metritis (also referred as toxic puerperal metritis or toxicmetritis) is characterized by an abnormally enlarged flaccid uterus anda fetid watery red-brown vulval discharge, associated with acute signsof systemic illness (mostly decreased milk yield, dullness or othersigns of toxemia) and fever >39.4° C. usually 10 days after parturition.

Endometritis involves clinical (acute) or sub-clinical inflammation ofthe uterine. Clinical endometritis is an inflammation of the endometriumonly, extending no deeper than the stratum spongiosum, and ischaracterized by a purulent vulval discharge, (typically up to 42 dayspost-partum) with no signs of systemic illness. Sub-clinicalendometritis may be diagnosed by the presence of more than 18%polymorphonuclear leukocytes in uterine cytology samples and/or byultrasonographic imaging of fluid in the uterine lumen collected 21-33days post partum, or the presence of more than 10% of polymorphonuclearleukocytes in uterine cytology samples at 34-47 days after parturation[Sheldon I M, (2006) ibid.].

Pyometra is the accumulation of significant fluid in the uterine lumenin the presence of a persistent corpus luteum and a closed cervix.

Any of the above may be caused by a variety of pathogenic bacteria. Incows, the causative organisms for metritis and endometritis are mostoften Arcanobacterium (Actinomyces) pyogenes, alone or in associationwith Fusobacterium necrophorum or other gram-negative anaerobicorganisms. In some forms of endometritis the causative agents areStaphylococcus hyicus or Escherichia coli. In other animals, such asmares, the most common etiologic agent of endometritis is Streptococcuszooepidemicus, but several other organisms may be involved, includingEscherichia coli, Pseudomonas aeruginosa, and Klebsiella pneumoniae.

Further, the term “uterine infection” encompasses infection of thecervix and of the vagina, such as, without being limited thereto,cervicitis, an inflammation of the cervix that may be caused by thebacterium Chlamydia trachomatis, by bacterium Neisseria gonorrhoeae(also called Gonococcus); or vaginitis, an inflammation of the vagina,that may be caused by the yeast Candida albicans, by the bacteriumGardnerella or by the parasitic protozoan Trichomonas vaginalis; andvulvitis, an inflammation of the vulva sometimes referred to asvulvovaginitis (because the vulva is also often inflamed when there isinflammation of the vagina).

In some dairy herds, 40% of the postpartum animals are diagnosed withuterine infections and thus are in need for veterinarian treatment(Lewis G. S., Uterine health and disorders. J. Dairy Sci., 80:984-994(1997)).

Cows diagnosed with uterine infections such as for example metritis orendometritis and retarded uterine and cervical involution, showsignificant reduction in conception rates, prolonged days to firstinsemination service as well as increased intervals from calving tobreeding which is exemplified by reduced numbers of pregnancies. Forexample, uterine infections may increase the period between calving tocalving, which optimally should not exceed an optimum period of about 12to 14 months, i.e. around one calving per cow per year. The ability tomaintain such an optimum period depends on numerous factors including,inter alia, the clinical state of the animal's uterus, which should becompletely free of any post-partum related infections.

Thus, uterine infections cause economic losses such as increased herdhealth-related costs, often reduce feed consumption, cause anappreciable reduction in milk production, and force breeders and milkproviders to cull livestock animals, such as cows, that would otherwisebe productive and remain in the herd.

Endometritis and metritis may be treated spontaneously and normallythere will be no appreciable effect on reproductive performance or anyother measure of productivity.

As appreciated, when a uterus becomes infected it may significantlyaffect milk production.

The invention is applicable for a variety of female mammalian animals,which may be either before a lactating stage (e.g. heifers) or alactating animal.

In some preferred embodiments, the female mammalian animal is anon-human mammalian. Possible non-human mammalian animals in the contextof the invention may be selected from the non-limiting group consistingof a dog, a sheep, a goat, a sow, a cow, a zebu, a zebra, a cat, adonkey, an ass, a buffalo, an oxen, a mare (including a filly), areindeer, a yak, a camel, a lama, an alpaca, an elephant, a pig, a boar,and a warthog. In one embodiment, the invention is applicable tolactating animals.

In yet another embodiment, the invention relates to livestock lactatinganimals, including, without being limited thereto, cows, buffalos,goats, sheep, mare, zebus, sows and oxen. A lactating animal ofparticular interest is a cow.

The mammalian animal may be any of nulliparous, primiparous ormultiparous lactating animal. A “nulliparous” (“nullipara” or “para”) isto be understood as referring to an animal that has never completed agestation period; a “primiparous” (“primipara” or “primip”) is to beunderstood as referring to an animal that has given birth to at leastone offspring (a single parturition); and a “multiparous” (“multip”) isto be understood as referring to an animal that has experienced two ormore parturitions.

In one embodiment, the uterine infection is at any stage before calving,e.g. in a heifer, as well as between two consecutive calvings.

In yet a further embodiment, the uterine infection is a post-partuminfection. In the context of the present invention, the termpost-partuminfection refers to infections occurring at any time after parturition,e.g. at the time interval between two consecutive parturitions and atsome embodiments, at a time after parturition until the beginning ofestrus. In some embodiments, the post partum period is considered to beduring the first 7 weeks after parturition.

The casein peptide is used to treat any of the above uterine infections.The term “treatment” or “treating” and the like are used herein to referto obtaining a desired pharmacological and physiological effect on thefemale mammalian animal having or in disposition of developing anuterine infection (e.g. being in post partum period). As such, theeffect may be prophylactic in terms of “preventing” or partiallypreventing an infection, symptoms or conditions thereof in a lactatinganimal in predisposition of developing an uterine infection and/or maybe therapeutic in terms of partial or complete cure of an alreadyexisting infection, condition, symptom or adverse effect attributed tothe infection. Thus, the term “treatment”, as used herein, covers any ofthe following (a) preventing the infection from occurring in a femalemammalian animal which may be predisposed to the infection (e.g. beforeas well as after calving) but has not yet been diagnosed as having it,e.g. the clinical symptoms of the infection have not yet being developedto a detectable level; (b) inhibiting the infection, i.e., arresting orreducing the development of the infection or its clinical symptoms suchas inflammation and/or fever; or (c) relieving the infection, i.e.,causing regression of the infection and/or its symptoms or conditions.

In some embodiments, treatment comprises parenteral (e.g. injection,infusion or the like) and/or topical administration of said at least onecasein peptide. The administration may be intrauterine and/or vaginaladministration. In one embodiment, the administration is transcervicalintrauterine administration.

Depending on the route of administration, the at least one caseinpeptide may be formulated with a suitable acceptable carrier to form apharmaceutical composition. An acceptable carrier is a pharmaceuticallyand physiologically acceptable carrier and is to be understood as acarrier or an excipient that is generally safe, non-toxic and neitherbiologically nor otherwise undesirable. A pharmaceutically acceptablecarrier as used in the specification and claims includes either one ormore than one of such carriers. The carrier can be any of thoseconventionally used and is limited only by chemico-physicalconsiderations, such as solubility and lack of reactivity with the atleast one casein peptide, and by the route of administidtion.

As appreciated by those versed in the art, treatment may also includecombined administration with other drugs, such as other antibiotics.

The pharmaceutical composition may also include other additive to giveform or consistency to the composition or to increase stability,sterility and isotonicity of the composition or to enhance uptake of thecasein peptide across membranes. For example, the additive may include apH adjusting agent, e.g. to maintain the pH of the composition in therange of 6 to 8, or even at times in the range of 6.7 to 6.9 which wasfound to be the most suitable pH for topical or parenteraladministration of casein peptides.

In one embodiment, the amount of the at least one casein peptideeffective to achieve treatment of uterine infection is between 10 ng/mlto 500 mg/ml per administration dose, at times, between 10 ng/ml to 200mg/ml per administration dose and even at times 10 ng/ml to 100 mg/mlper administration dose. In some embodiments, the casein peptide is acasein hydrolyste comprising a mixture of casein peptides, at least aportion of which being therapeutically effective to treat the uterineinfection. At times, the casein peptide may be administered in anon-solution form (e.g. cream), in which case, the amount will bebetween about 10 ng/kg body weight to 200 mg/kg body weight peradministration dose, at times, between about 10 ng/kg body weight to 100mg/kg body weight per administration dose. An administration dose refersto a single administration event, i.e. an animal may receive a singleadministration dose or several administration doses per day, or duringtwo or more days.

In one embodiment, the at least one casein peptide is administered once(single dose administration). In some other embodiments, two or moreadministrations of said at least one casein peptide are executed,typically between 2 to 6 doses. When more than one dose of the at leastone peptide is administered, it is typically (albeit not limitedthereto) that a time interval of from 2 hours to 14 days is used betweentwo consecutive administrations. Notwithstanding the number ofadministrations, it is in accordance with one embodiment that the singleor first administration is after calving, typically up to 120 days postcalving, more particularly, up to 70 days post calving, further at timesup to 30 days post calving, and at times immediately post calving (i.e.minutes or hours after calving) as well as up to day 26 post calving.

As used herein, the forms “a”, “an” and “the” include singular as wellas plural references unless the context clearly dictates otherwise. Forexample, the term “a casein peptide” includes one or more such peptides,e.g. mixture of peptides which are effective in treating uterineinfection.

Further, as used herein, the term “comprising” is intended to mean thatthe composition include the recited one or more peptides, i.e. the atleast one casein peptide (which may by itself relate to a preparationcomprising a mixture of such peptides), but not excluding otherelements, such as physiologically acceptable carriers and excipients aswell as other active agents. The term “consisting essentially of” isused to define compositions which include the recited elements butexclude other elements that may have an essential significance ontreatment of uterine infection. “Consisting of” shall thus meanexcluding more than trace elements of other elements. Embodimentsdefined by each of these transition terms are within the scope of thisinvention.

Further, all numerical values, e.g. when referring the amounts or rangesof the elements constituting the composition comprising the at least onecasein peptide as an active ingredient, are approximations which arevaried (+) or (−) by up to 20%, at times by up to 10% of the statedvalues. It is to be understood, even if not always explicitly statedthat all numerical designations are preceded by the term “about”.

The invention will now be exemplified in the following description ofexperiments that are carried out in accordance with the invention. It isto be understood that these examples are intended to be in the nature ofillustration rather than of limitation. Obviously, many modificationsand variations of these examples are possible in light of the aboveteaching. It is therefore, to be understood that within the scope of theappended claims, the invention may be practiced otherwise, in a myriadof possible ways, than as specifically described hereinbelow.

The following examples are presented in order to more fully illustratethe preferred embodiments of the invention. They should in no way beconstrued, however, as limiting the broad scope of the invention sincenumerous modifications and variations therein will be apparent to thoseskilled in the art.

DESCRIPTION OF SOME NON-LIMITING EXAMPLES

Materials

Propidium iodide (solution for fluorescence, CAS 25535-16-4,Sigma-Aldrich catalogue 70335, Sigma-Aldrich Co., 3300 South 2nd Street,St. Louis, Mo. 63118).

Recombinant human IL-8 (rhIL-8) (ProSpec-Tany TechnoGene Ltd., RehovotScience Park, P.O. Box 398, Rehovot 76103, Israel).

Isotonic Percoll (based Percoll® Sigma-Aldrich catalogue P4937,Sigma-Aldrich Co., 3300 South 2nd Street, St. Louis, Mo. 63118).

Non-viable Streptococcus zooepidemicus suspension (Omnisorbin,Calbiochem, Bad Soden, Germany).

Oxytetracycline dehydrate 200 mg/mL (Alamycim®, Norbrook Laboratory Ltd,UK).

Water for irrigation (Cure Medical, Teva Medical, Israel). In thefollowing, also, the cows are Israeli Holstein-Friesian dairy cows.

Phorbol myristate acetate (PMA, Sigma-Aldrich catalogue P8139,Sigma-Aldrich Co., 3300 South 2nd Street, St. Louis, Mo. 63118).

Nonfluorescent dye dihydrorhodamin 123 (DHR 123, Morbitec, Gottingen,Germany).

Example 1 Preparation of Casein hydrolysate

Preparation of Stock Solutions: 25 mM Tris HCl at pH=8.0 (20 liters) and25 mM Tris HCl at pH=7.0 (5 liters).

Tris base (Applichem GmBH, Darmstadt, Germany) was weighted (75.687 gr)and dissolved in a glass bottle equipped with magnetic stirring barcontaining 24 liters of water for irrigation (WFI, Cure Medical,manufactured by Teva Medical, Israel), at a final pH of about 11.5,until clarification.

To the 19 liter solution, the pH was adjusted to about 7.5-8.5(preferably about 8) using 10N HCl (Sigma-Aldrich, Israel) and thevolume was finalized to 20 liter by adding WFI. To the 4.75 litersolution, the pH was adjusted to between about 6.75-7.25 (preferably7.0) and the final volume was finalized to 5 liter by adding WFI. Thesolutions were stored at room temperature until further use.

Preparation of Casein-Derived Peptides:

Casein hydrolysate was prepared by Biological Industries Israel (BeitHaemek Ltd., Israel) according to the following:

Tris HCl 25 mM, at pH=8.0 (20 liters) were added into a glass bottle andthe temperature was adjusted to between about 40° C. to 48° C.,preferably 45° C.

A total of 4,000 gr sodium caseinate (DMV International.FrieslandCampina Central Office, Amersfoort, The Netherlands) were addedinto the Tris HCl solution to obtain a 20% (w/v) solution. The mixturewas stirred gently to reachfull dissolution. The solution pH wasmaintained at about 8.0 and the temperature at about 45° C. Then 4 g ofporcine trypsin powder (Sigma-Aldrich, Israel) were added to thesolution.

The mixture was gently stirred on a magnetic stirrer or deflocculating(preferred) stirrer under gentle agitation at about 45° C. Thehydrolysis was allowed to proceed for a total of about one hour. The pHwas then adjusted to about pH 8.0 using 10N NaOH (JT Baker, AvantorPerformance Materials, Phillipsburg, N.J., U.S.A) and the temperaturewas maintained at about 45° C.

The trypsin in the crude hydrolysate was inactivated by elevating thetemperature to 90° C.-95° C. for one hour. The supernatant was cooled toroom-temperature by using ice in an external bath. The pH was adjustedto 4.7 using 10N HCl. After 15-30 minutes, the insoluble material wasremoved by centrifugation for 20 min, at 2-8° C., 4500 rpm.

The supernatant was transferred to a new sterile glass bottle and the pHwas adjusted to 7.0 using 10N NaOH.

The solution volume was adjusted to 20 liters by adding the 25 mM TrisHCl solution at pH 7.0.

The final volume was filtered sequentially through 1.2 μm/0.65 μmpre-filters and through two 0.45/0.22 μm sterile CA membrane filtercapsules. The filtered solution was transferred to glass serum bottle(100 ml and 200 ml respectively) by aseptic filling in sterile bench.The samples were stored at −20° C. (frozen) until further use.

The product (raw material) comprised 20% Sodium Caseinate, 25 mMTris-HCl and 0.02% Trypsin.

Example 2 Clinical Study 1—Low Dose Studies of the Effect of CaseinHydrolysate in Acute Post-Partum Bovine Clinical-Metritis

The effects of bovine casein hydrolysate in comparison with antibioticdrugs, was studied in dairy cows suffering from acute puerperal metritis(Sheldon et al., ibid 2006).

General Study information:

The cows recruited for this study were lactating Holstein Friesianlocated in large dairy herd in Israel (longitude 34° 40′ E-35° 30′E;latitude 30° 30N-33° 01′N). The cows were milked 3 times a day. The herdused was free-stall barn with a size ranged from 300 to 350 milkingcows.

At dairy farm an administrating veterinarian was selected and appointedby the Clinical Research Organization (CRO) (Hachaldait-Israeli CattleBreeders Association, Caesaria Industrial Park, Israel) in order tocarry out the study functions, specifically the administratingveterinarian was responsible for treatment administration, clinicalassessment, training dairy herd personal, proper storage, performnecessary clinical observations for adverse event assessment and fillreport forms. All study documents were filled out legibly in indelibleblack/blue ink. The original data were sent to the sponsor.

Physical Observations:

All dairy cows were routinely examined in the first 5 to 10 dayspostpartum and their physical condition was assessed using measures of:

(i) status of the uterus by trans-rectal palpation;

(ii) heart girth measurements for approximate weight determination;

(iii) a body condition score provided by the veterinarian, which isbased on the cow's height and fat composition in various parts of thebody according to Edmonson A. J., et al. [Edmonson A. J., Lean I. J.,Weaver L. D., Farver T., Webster G. A body condition scoring chart forHolstein dairy cows. J. Dairy Sci., (1989); 72:68-78].

Cows are enrolled to the study provided that their physical condition iscomplied with the inclusion criteria as follows:

-   -   Cows with clinical postpartum metritis,    -   Cows with non-twins delivery,    -   Cows were not administered with intrauterine or systemic        reproductive hormone therapy during the last 4 weeks,    -   Cows with no diagnosed morbidities, which may be potentially        influence the outcome of treatment in the judgment of the        investigator,    -   The herd managers agreed not to administer any intrauterine or        reproductive hormonal therapy before examination of cows for        this trial.

In addition, cows were enrolled to the clinical study provided that theyhave none of the following characteristics (herein “exclusioncriteria”):

-   -   Cows had abnormal internal genitalia (including adhesions)    -   Cows with diagnosis and or treatment of severe vaginitis,        lameness, mastitis-ketosis and digestive disorders during the        current post calving period or any other clinical disease        requiring systemic treatment at enrollment,    -   Cows identified to be culled (not to be inseminated) after        calving    -   Cows administrated with immunotherapy within 3 weeks prior to        study entry,    -   Cows administrated with anti-inflammatory and/or anabolic        therapies, either systemic or by feeding 2 weeks prior to study        entry.    -   Cows which are suspected or with a diagnosis of retained fetal        membranes at the time of the current calving.    -   Cows which are suspected or with a clinical evidence of acute        puerperal metritis with a systemic effect    -   Cows which are indented to be removed for any reason from the        farm in the current location.

Cows in a proper physical condition (as defined above in the inclusioncriteria and the exclusion criteria), that were diagnosed with confirmedclinical metritis, as determined by any one of the following clinicalcriteria, were enrolled to this clinical study:

1) cows must be febrile (i.e., morning rectal temperature more than39.4° C.) and/or

2) cows must have a flaccid, non-retractable uterus that is located inthe abdomen, a cervical diameter >7.5 cm, and a watery, mucopurulent, orfetid vulvar discharge.

Study Specifics:

On day “0” (first day of the study), dairy heifers or primiparous ormultiparous dairy cows from the same herd and within the same lactationand ages were randomly divided, using tables prepared in advance by theinvestigator, to receive one or other of the treatments. Within eachtable, cows were assigned to treatment in pairs, a cow for each group.

Treatment:

Case study Arm (“arm 1”)—450 mg of bovine casine hydrolysate (BatchNumber B100) administered in a 150 ml solution until completion of thearm's sample size.

Control Arm (“arm 2”)—25 ml of Oxytetracycline dehydrate (200 mg/mltotal of 5,000 mg) (Norbrook Laboratory, UK).

No other intrauterine treatments were provided to the tested cows(including but not limited to intrauterine antimicrobials, internal orexternal teat systemic antimicrobials).

Treatment was initiated at day “0” and was administered to the cowsthrough transcervical intrauterine infusion using a sterile uterinecatheter.

All the experiments were blind case-control studies as all unidentifiedadministrated products were labeled in vials of bovine caseinhydrolysate and the negative control was always administered to cows bythe same route as the treatment, thus the investigators or herdspersonsor veterinarian was unable to identify the study cases or the controls.

Study Endpoints:

The end points of this study were safety and efficacy as detailed below.

Safety:

-   -   Determining maximum severity of any toxicity probably related to        bovine casein hydrolysate with the current dose.    -   Determining maximum severity of any toxicity possibly related to        bovine casein hydrolysate with the current dose.

Safety was determined by monitoring on days 0.3, 1, 2, 3, 7 and 14post-treatment, the appearance of adverse events (as detailed below) orany sign of toxicity.

In addition, the overall general impression from each cow was assessedand ranked according to the description on Table 1.

TABLE 1 Ranking of cow's general impression Score Description 0 Normal 1Slightly depressed 2 Depressed (solitude, head leaning down)

The program manager/monitor/sponsor was responsible to immediatelynotify on any possible signs of AEs. Appropriate actions may involvelocating and breaking blinding codes so that appropriate medicaltreatment can be given, recording of the AEs in the study documentation,and reporting AEs to the sponsor.

According to veterinary criteria if adverse events were encountered(including in the lapse time of the first post treatment visit) orsignificant signs and symptoms of clinical post-partum metritis wereobserved at day 7 post treatment, same treatment as provided in day 0was administrated to the cow.

All serious and/or unexpected adverse events were documented.

Efficacy:

Treatment success was measured by the average reduction of the followingtwo parameters:

-   -   if the animal group had an average rectal temperature reduction        at day 14 post treatment compared to the average temperature        measured during day 0 and/or;    -   if the animal group had an average reduction in the discharge        parameters at day 14 post treatment.

Efficacy was assessed by measuring the rectal temperature; genitalsystem morphology and the genital secretions at a first visit after themorning observation and milking, before the treatment, 6-8 hours afterthe treatment and then on days 1, 2, 3, 7 and 14 post treatment. Ingeneral, treatment success was assessed after a total of 14 days aftertreatment.

The administrated veterinarian examined all cows and was in charge onthe ranking of the genital secretions, from 1 to 4, based on thedescriptions provided in Table 2.

TABLE 2 Ranking of genital system secretion Score Description 0 None. 1Transparent thin secretion 2 Thin serosanguinotic secretion 3Mucopurulent secretion 4 Purulent secretion with foul smell

The genital system morphology was assessed and ranked according to thedescription in Table 3.

TABLE 3 Ranking of genital system morphology Score Description 0 Normalappearance of the vulva and vagina, no apparent redness, swelling, signof local pain or signs of inflammation 1 Mild redness and swelling 2Severe serosanguinotic secretion, swelling and/or injuriesPost Treatment Cow Management

Generally, post-treatment animal management and observation scheduleswere be followed according to the specific facility's practice.Following treatment, study cows have been commingled with non-study cowsand will be housed in accordance with each site's standard practices.All cows have been subjected to the same basic management (includinghousing and rations, the timing of changes in housing or ration, and anyvaccination protocols, exclusive of vaccination against metritispathogens if such are available

Preparation and Storage of Materials for the Study:

Bovine Casein Hydrolysate:

-   -   After manufacture, bovine casein hydrolysate was immediately        frozen to reach a temperature between −15 to −20° C. This        process was done in less than 16 hours from manufacture process.        As described above, 155 ml casein hydrolysate was filled in        vials of 200 ml, out of which 150 ml were injected (the rest 5        ml were kept for follow up).    -   Package vials containing the bovine casein hydrolysate were        transported from the manufacture facility to the participating        dairy herds through Cold Transport Delivery (Frozen) and were        stored at each dairy herd in a designated freezer (−20° C.)    -   One day before (24-36 hours) the scheduled treatment day (after        the morning milking), vials containing the biologic products        were taken out of the freezer and kept in a cooling        refrigerator's temperature (4-8° C.) until the time of        administration.    -   At any case that the content of a vial containing the bovine        casein hydrolysate was not administrated, the vial was stored in        the original package, marked 000, and was not administrated at        any time.

Antibiotics were kept refrigerated (4-8° C.), to avoid prolongedexposure to moisture and direct sunlight.

Results—Clinical study 1—Low Dose Study

Randomized blind safety studies were conducted on a total of 16 cows,eight cows were administered with casein hydrolysate (treatment, arm 1)and eight cows were administered with antibiotics (control, arm 2).

All tested cows had a normal behavior during the 14 days post treatment,specifically the general impression from each cow was ranked as “0” inTable 1 following treatment administration with none of the cows showingany signs of depression, less food intake.

Treatment efficacy was measured, at the indicated times after thetreatment administration, by measuring the fever of each cow and bymonitoring the genital system secretion and morphology. The secretionsand morphology for each cow were ranked according to the description inTable 2 and in Table 3, respectively.

Comparison of the results obtained from cows treated with caseinhydrolyasate (150 ml) and from cows treated with antibiotics indicatedthat casein was able to maintain the same body temperature asantibiotics (Table 4).

TABLE 4 Effect of casein on the cow's fever Bovine Casein HydrolysateAntibiotics Time of measurement (° C.) (° C.) Before treatment (day = 0)38.8 ± 0.57 38.8 ± 0.26 6-8 h after treatment 39.4 ± 0.54 39.4 ± 0.66Day 1 (22-32 h after 39.0 ± 0.49 39.1 ± 0.27 treatment) Day 2 39.2 ±0.40 39.1 ± 0.47 Day 3 38.8 ± 0.42 38.9 ± 0.37 Day 7 38.8 ± 0.44 38.9 ±0.18 Day 14 38.8 ± 0.33 38.8 ± 0.41

The genital system morphology of all the treated cows was found to benormal, and ranked “0” with no appearance of redness or swelling asdescribed in Table 3, meaning there were no local adverse events.

In addition, it was observed that the treatment of the cows with caseinhydrolysate for 14 days reduced the genital secretions by about 50%similarly as antibiotics (Table 5).

TABLE 5 Effect of casein on genital secretions Bovine Casein Time ofHydrolysate Antibiotics measurement (ranking) (ranking) Before treatment3.875 ± 0.353  3.625 ± 0.517  (day = 0) 6-8 h after treatment 3.75 ±0.462 3.0 ± 0.92 Day 1 (22-32 h after 3.75 ± 0.462 2.12 ± 0.84 treatment) Day 2 3.25 ± 1.03  2.0 ± 0.92 Day 3 3.25 ± 1.03  2.0 ± 0.92Day 7 3.0 ± 0.92 1.5 ± 0.75 Day 14 2.0 ± 1.09 1.5 ± 0.75

The results described above and relating to Tables 1 to 5 clearly showthe following:

-   -   administration of bovine casein hydrolysate to cows at a dose of        450 mg, was not toxic and did not cause any local adverse        effects and systemic adverse effect and therefore may be safely        intrauterine administered to cows.    -   In terms of efficacy, as shown in the fever measurements as well        as in the genital morphology and genital secretions        measurements, administration of 450 mg bovine casein hydrolysate        was found to be as efficient as the common metritis treatment,        antibiotics, in treating postpartum clinical metritis.

The above results provide evidence for the beneficial therapeuticadvantage of using casein peptide over standard antibiotics, at leastfor the following reasons: casein is a natural material against whichresistance would typically not be developed (as opposed to antibiotics),and further, since casein is a natural material the milk milked from theanimal during casein peptide treatment may be used (and not discarded,as done with antibiotics-treated lactating animals).

Example 3 Clinical Study 2—High Dose Studies of the Effect of CaseinHydrolysate in Acute Post-Partum Bovine Clinical-Metritis

The cows recruited for this study were lactating Israel HolsteinFriesian located in a large dairy herd in Israel (longitude 34° 40′E-35° 30′E; latitude 30° 30′N-33° 01′N).

Cows enrollment to the study with respect to the physical conditions,inclusion criteria and exclusion criteria were as detailed above forclinical study −1 with respect to the overall physical condition andwith respect to the diagnosis with clinical post partum metritis. Inaddition, cows randomization was as detailed above.

On day “0” (first day of the study) dairy heifers or primiparous ormultiparous dairy cows from the same herd and with the same lactationand ages were randomly divided, using tables prepared in advance by theinvestigator, to receive one or other of the treatments. Within eachtable, cows were assigned to treatment in pairs, a cow for each group.

Treatment:

Case study Arm (“arm 1”)—1350 mg of bovine casein hydrolysate (BatchNumber B100) administered in a 150 ml solution 3 times.

Control Arm (“arm 2”)—25 ml of Oxytetracycline dehydrate (200 mg/mltotal of 5,000 mg) (Norbrook Laboratory, UK).

No other intrauterine treatments were provided to the tested cows(including but not limited to intrauterine antimicrobials, internal orexternal teat systemic antimicrobials).

Treatment was initiated at day “0” and was administered to the cowsthrough transcervical intrauterine infusion using a sterile uterinecatheter.

All the experiments were blind case-control studies as all unidentifiedadministrated products were labeled in vials of bovine caseinhydrolysate and the negative control was always administered to cows bythe same route as the treatment, thus the investigators or herdspersonsor veterinarian was unable to identify the study cases or the controls.

Study Endpoints:

Safety and Efficacy Criteria are as Detailed Above for Clinical Study 1(Example 2)

The administrated veterinarian examined all cows and was in charge onthe ranking of the genital secretions, from 1 to 4, based on thedescriptions provided in Table 2 of clinical study 1 as well as thegenital system morphology according to the description in Table 3 ofclinical study 1 and the overall general impression from each cowaccording to the description on Table 3 of clinical study 1.

In addition, the veterinarian aseptically will prepare the genital area,specifically the vulva to perform uterine swabs's samples and to obtainuterine cytology samples from the designated cows. Uterine swab samplesare taken from the uterine body and are obtained by using protectionsterile uterine swabs.

Uterine swab samples for uterine cytology samples are obtained beforethe treatment and on days 7 and 14 post treatments.

Prior to swab sampling, labeling of samples' slide is done and includesCow ID and indication, date of indication, Site ID and Date and Time ofSampling. Additional submission forms are filled according to laboratoryinstructions.

Swabs after being sampled are rolled on a designated glass slide in aclean environment. The rolled process starts from the label side (white)to the end of the slide and is repeated twice or three times. Biologicalsamples should not be included in both extremes parts of the slide. Eachsample slide is immediately placed separately in a transport medium andtransfer to the laboratory at 4° C.-8° C. within three (3) hours aftercollection. Immediately upon receipt, uterine swabs are re-suspended in2 ml thioglycolic broth that is partly diluted 100 times by adding morebroth.

All uterine samples are analyzed for pathology, for example, for thepresence of neutrophils, lymphocytes, basophiles, macrophages,eosinophils.

Also, on day 14, the presence of infection after uterine swabs sampleperformed during pre-treatment and post-treatment (day 14) is monitored.

In addition uterine swab sample are performed after 7 and 14 days asdescribed above and on day 14, assessment of any adverse event was doneas detailed above.

Efficacy in this experiment is measured by parameters associated withthe ability to prevent uterine infections in non-retained fetal membraneanimal as follows:

(i) Microorganism parameters: clinical and bacteriological negativerates after treatment compared to control (main parameters). Generally,bacteria is categorized, according to known pathogenicity within theuterus (Sheldon I. M., Noakes D. E., Rycroft A. N., Dobsons H. Effect ofintrauterine administration of oestradiol on postpartum uterinebacterial infection in cattle. Animal Reproduction Science, 81:13-23(2004)). The categories vary from 1 to 3 based in clinical endometritis,which correspond to:

Category 1—bacteria that frequently cause metritis;

Category 2—bacteria that are an infrequent cause of metritis; and

Category 3—bacteria not recognized as uterine pathogens.

Bacterial growth scores are summed-up for all bacteria (total bacterialscore) or for each of the pathogen categories (1-3).

(ii) Biological status: by measuring artificial inseminationperformance, pregnancy hazard and milk yield (main parameters).

According to Veterinary criteria if adverse event were encountered(including in the lapse time of the first post treatment visit) orsignificant signs and symptoms of clinical post-partum metritis, sametreatment was administrated to the cow (bovine casein hydrolysate tocases and antibiotics to controls). All study documents were filled outlegibly in indelible black/blue ink. The original data were sent to thesponsor.

Post treatment management was as detailed above in clinical study 1.

The materials were prepared and stored as detailed above in clinicalstudy 1.

Results—Clinical study 2—Efficacy Study:

Randomized blind safety studies were conducted on a total of 7 cows, 4cows were administered with casein hydrolysate (treatment, arm 1) and 3cows were administered with antibiotics (control, arm 2).

No adverse effects were observed in any of the cows treated for 14 dayswith casein hydrolysate, specifically the general impression from eachcow indicated that the cow behavior was normal during the 14 days posttreatment, specifically the general impression from each cow was rankedas “0” in Table 1, following treatment administration, with none of thecows showing any signs of depression or less food intake.

The genital system morphology of all the treated cows was found to benormal, and ranked “0” with no appearance of redness or swelling asdescribed in Table 3 in clinical study 1 above, meaning there were nolocal adverse events.

Comparison of the results obtained from cows treated with caseinhydrolyasate (450 ml) and from cows treated with antibiotics indicatedthat casein was able to prevent increase in body temperature to anextent similar to that obtained with antibiotics (Table 6).

TABLE 6 Effect of casein on the cow's fever Casein HydrolysateAntibiotics Time of measurement (° C.) (° C.) Before treatment 38.9 38.4(day = 0) 6-8 h after treatment 38.6 39.1 Day 1 (22-32 h after 38.7 38.6treatment) Day 2 38.1 38.4 Day 3 38.4 38.9 Day 7 38.6 38.3 Day 14 38.838.7

The results show that treatment of casein hydrolysate is effective,similar to antibiotics and no adverse events were observed, even at highdose treatment.

Examinations, Inclusion and Exclusion Criteria for Examples 4-8

Routine Examination of the Cows

Dairy cows are routinely examined immediately after calving to assesstheir physical conditions using measures of:

(i) status of the uterus by trans-rectal palpation and ultrasonograph;

(ii) heart girth measurements for approximate weight determination;

(iii) a body condition score provided by the Veterinarian, which isbased on the cow's height and fat composition in various parts of thebody according to Edmonson A. J., et al. (Edmonson A. J., Lean U.,Weaver L. D., Farver T., Webster G. A body condition scoring chart forHolstein dairy cows. J. Dairy Sci., 72:68-78 (1989).

Cows which are diagnosed with postpartum health abnormalities, forexample ketosis, retained placenta, left displays abomasums, lameness,are provided with appropriate medical treatment and are excluded fromany future study trial.

General Examination Protocol (Examples 4-8):

In all in vivo experiments described below, the tested populationincludes about 25% dairy heifers or primiparous and about 75%multiparous dairy cows (at post partum period). Dairy cows from the sameherd and with the same lactation and age are randomly distributed withinthe different study groups. In addition, all experiments are blindcase-control studies as all unidentified products to be administratedare labeled in vials of bovine casein hydrolysate and the negativecontrol is always administered to cows by the same route as thetreatment, thus the investigators or herdspersons is unable to identifythe study cases or the controls. Unless otherwise indicated alltreatments (including saline) are administered by transcervicalintrauterine infusion.

Cows and heifer calving during the study period will be included in thistrial. All cows will be examined with respect to following inclusioncriteria and exclusion criteria.

The inclusion criteria for participation in the study are:

-   -   Cows with a normal health uteri (only for example numbers 4 and        5),    -   Cows with a uterine infection (for examples 6, 7 and 8),    -   Cows with non-twins delivery,    -   Cows not being administered with intrauterine or systemic        reproductive hormone therapy in the current lactation,    -   Cows not diagnosed with morbidities, which may be considered to        potentially influence the outcome of treatment in the judgment        of the investigator,    -   Herd managers agreed not to administer any intrauterine or        reproductive hormonal therapy before examination of cows for        this trial.        The exclusion criteria for not participation in this study are:    -   Cows with an abnormal internal genitalia (including adhesions),    -   Cows diagnosed with and or treated to severe vaginitis, retained        fetal membrane history, lameness, mastitis, ketosis and        digestive disorders during the current post calving period or        any other clinical disease requiring systemic treatment at        enrollment,    -   Cows identified to be culled (not to be inseminated) after        calving,    -   Cows administered with immunotherapy within 3 weeks prior to        study entry,    -   Cows administered with anti-inflammatory and anabolic therapies,        either systemic or by feeding within 2 weeks prior to study        entry.

Example 4 Clinical Study 3—Studies of the Effect of Bovine CaseinHydrolysate in Reproductive Performance of Dairy Cows

The goal of this study is to evaluate the effect of bovine caseinhydrolysate treatment post partum on reproductive performance of dairycows; the effect of intra uterin infusion of casein hydrolysate on thecow's reproductive performance which will be compared to control.

Study will be considered successful provided that at an average periodof 49-70 days postpartum, one or both of the following criteria isachieved in the bovine casein hydrolysate treated cows:

(1) the artificial insemination submission rate is at least 5% higher,preferably 10% higher, most preferably 20% higher compared to controls;and/or

(2) the open days (calve to breeding) are shorter at about 5-9 days,preferably 10 days or more compared to controls or that a statisticallysignificant result is achieved in the difference of the open daysbetween cases and controls Following the clinical examination, recruiteddairy cows will be blinded assigned within herd after calving to thedifferent study groups, i.e. bovine casein hydrolysate treated cows andcontrol cows. Cows will be randomly assigned to the two groups.

Treatment:

-   Treated cows—will be administered by an i.u infusion bovine casein    hydrolysate.-   Control cows—will be administered by an i.u infusion control    solution

Each animal will be reexamined biweekly after recruitment similar as atenrollment up to day 40-60 post calving.

After the voluntary waiting period, of about 42 days, cows will besubjected to a synchronization of ovulation and timed inseminationprotocol for the first service. Pregnancy diagnosis will be conducted bypalpation per rectum of the uterus and its contents at about 42 to 49 dafter insemination.

Insemination dates will be collected using on-farm data-recording formsand data records. Reproductive performance on individual animals will becollected for a minimum of 2 months after enrollment. Observations oftime to pregnancy will be censored.

The following descriptive outcomes will be measured to assessreproductive performance:

-   -   interval from calving to first insemination (days to first        service),    -   first service pregnancy (%),    -   interval from calving to pregnancy (day open),    -   number of inseminations for pregnant cows,    -   removal risk for reproductive failure (%),    -   milk yield (liters per day),

Observations of time to pregnancy for cows that will be culled duringthe trial before pregnancy will be censored on the date of culling. Forcows that will be not pregnant at the termination of data collection,observations will be censored on the last date.

Example 5 In Vivo Prophylaxis of Postpartum Uterine Infections in DairyCows

The effects of bovine casein hydrolysate on systemic preventivetreatment of metritis and endometritis (clinical and subclinical) willbe evaluated in dairy cattle (Sheldon I. M., Lewis G. S., LeBlanc S.,Gilbert R. O. Defining postpartum uterine disease in cattle.Theriogenology, 65:1516-1530 (2006)).

The goal of this study is to evaluate the efficacy of a preventivetreatment of cows from uteri infectious with an administration of bovinecasein hydrolysate. Specifically, to prevent metritis and endometritis(clinical and subclinical) after an intrauterine infusion of bovinecasein hydrolysate in cows, administered after calving regardless oftheir body temperature.

The objective of this study is to prevent the occurrence of uterineinfection or to minimize its effects after it has occurred after ablanket preventive treatment to be carried post-partum by intra uterineinfusion of bovine casein hydrolysate in dairy cows and heifers withoutretained fetal membrane compared to controls.

During enrollment the following examinations will be held:

(i) a general physical examination and a clinical examination performedon the reproductive tract by transrectal palpation as well as rectalfever measurements, respiratory evaluation and heart beat measurements.

(ii) uterine bacteriology examination—uterine bacteriology samples cellsare obtained for determination of endometrial bacteriological status bythe presence of microorganism (infections). After calving, the cows areassigned into two study groups. a bovine casein hydrolysate(administered by intra uterine infusion) (Group I), and control (GroupII).

In addition, for all clinical evaluations, the treatment is performedalso in cows without retained fetal membrane and regardless of the cow'sbody temperature.

Treatment follow up is performed on biweekly basis examinationsincluding similar examinations as in the enrollment—treatment stage.Finally, on day 42 post treatment, endometrial (bacteriology)examination as well as clinical examination are performed to evaluatethe presence or absence of uterine infections.

Efficacy in this experiment is measured by parameters associated withthe ability to prevent uterine infections in non-retained fetal membraneanimal as follows:

(i) Microorganism parameters: clinical and bacteriological negativerates after treatment compared to control (main parameters). Generally,bacteria are categorized, according to known pathogenicity within theuterus (Sheldon I. M., Noakes D. E., Rycroft A. N., Dobsons H. Effect ofintrauterine administration of oestradiol on postpartum uterinebacterial infection in cattle. Animal Reproduction Science, 81:13-23(2004)). The categories vary from 1 to 3 based in clinical endometritis,which correspond to:

Category 1—bacteria that frequently cause metritis;

Category 2—bacteria that are an infrequent cause of metritis; and

Category 3—bacteria not recognized as uterine pathogens.

Bacterial growth scores are summed-up for all bacteria (total bacterialscore) or for each of the pathogen categories (1-3).

(ii) Biological status: by measuring artificial inseminationperformance, pregnancy hazard and milk yield (main parameters).

Example 6 In vivo Effects of Casein Hydrolysate in Clinical Endometritis

The effect of bovine casein hydrolysate in comparison with control isevaluated in dairy cows suffering from clinical endometritis, in theirpostpartum period.

A routine examination of all cows in the herd within about 26 to 33 dayspostpartum includes a general physical examination and a clinicalexamination performed on the reproductive tract by transrectal palpationas well as rectal fever measurements, respiratory evaluation and heartbeat measurements. Cows and heifers which are diagnosed with clinicalendometritis based on clinical and bacteriology criteria which includesfever, typically high fever, bad smell, the presence ofpolymorphonuclear neutrophil granulocytes and microorganism/bacteria inendometrial (Sheldon et al., ibid 2006), but without previously beingdiagnosed with puerperal metritis, are recruited to the study.

The studied cows and/or heifer are assigned into two study groups abovine casein hydrolysate (administered by intra uterine infusion)(Group I), and control (Group II).

Seven days post treatment (between about 33 and 40 days postpartum),each cow is clinically, cytology and bacteriology examined as in theenrollment. Animals which are still diagnosed with clinical endometritisand therefore are not clinically cured, receive a second dose of thesame treatment as initially administered. These animals are thenexamined for a third and final time after additional 14 days (betweenabout 47 and 54 days post partum; 21 days after initial enrollment).

Efficacy may be considered in the case groups with a clinical cure ratehigher than 60% or a statistically significant result. The effect oncure rate will be determined primarily by changes in the bacteriologicaltest results—i.e. the presence of no microorganism according to Sheldonet al criteria (Sheldon et al ibid 2004) after at least onebacteriological test during the 7 to 9 days after treatment and nulldischarge detectable in the vagina.

Clinical bacteriology cure may be classified based on the followingculture results (Sheldon et al ibid 2004).

-   -   (i) Treatment success=cultures positive at clinical endometritis        diagnosis and cultures negative after 7 days post treatment from        the same microorganism which frequently cause metritis,    -   (ii) Treatment failure=cultures positive for the same organism        at clinical endometritis which frequently cause metritis on        diagnosis day and 7 days post treatment,    -   (iii) Inconsistent result=culture positive at clinical        endometritis on diagnosis day and 7 days post treatment but with        different organisms which frequently cause metritis.

Example 7 In Vivo Effect of Casein Hydrolysate in Sub-ClinicalEndometritis

The effects of bovine casein hydrolysate are evaluated in dairy cowssuffering from sub-clinical endometritis (Sheldon et al., ibid 2006).

A routine examination of all cows in the herd within about 26 to 33 dayspostpartum includes a general physical examination and a clinicalexamination performed on the reproductive tract by transrectal palpationas well as rectal fever measurements, respiratory evaluation and heartbeat measurements. Cows and heifers which are diagnosed withsub-clinical endometritis based on clinical and cytology criteria whichincludes the presence of >18% polymorphonuclear neutrophil granulocytesin uterine cytology samples collected about 26-33 days postpartum, arerecruited to the study.

The studied cows and/or heifer are assigned into two study groups abovine casein hydrolysate (administered by intra uterine infusion)(Group I), and control (Group H). After 14 days post treatment (betweenabout 40 and 47 days postpartum) each cow, case and controls, isclinically and cytology examined as in the enrollment. Animals stilldiagnosed with sub-clinical endometritis and therefore not clinicallycured receive a second dose of bovine casein hydrolysate as initiallyadministered or as specified by a veterinarian.

Efficacy maybe considered in the case groups with a clinical cure ratehigher than 60% or a statistically significant result, and/ormeasurement of less than 15% of polymorphonuclear neutrophilgranulocytes or a statistically significant difference between cases andcontrols at the endometrial cytology examination, and/or biologicalstatus by measurement artificial insemination performance and/orpregnancy hazard Clinical and cytology cure rate will be determinedprimarily by the cytology parameters.

Example 8 In Vivo Effect of Casein Hydrolysate in Acute PuerperalMetritis

The effects of bovine casein hydrolysate in comparison with control willbe studied in dairy cows suffering from acute puerperal metritis(Sheldon et al., ibid 2006).

Following a routine examination of all cattle through about 7 to 14 daysafter calving, cattle which will be diagnosed with confirmed puerperalmetritis according to the following clinical criteria, are enrolled tothis study:

1) cows must be febrile (i.e., morning rectal temperature more than39.5° C.-103.10° Fahrenheit), and

2) cows must have a flaccid, non-retractable uterus that is located inthe abdomen, and a watery, fetid vulvar discharge.

On the diagnosis day the tested cows are assigned into two study groups.Each group is administered twice a week by an interval of 48 hours withthe following treatments of either bovine casein hydrolysate (Group I),or cephapirin benzathine (Group II).

Efficacy is assessed by comparing the examination of the clinicalsymptoms, bacteriology results and rectal temperature after enrollment,at first day of treatment and in each one of the consecutive about 7 to14 days post treatment. Cows are observed with rectal temperatures takenafter the animal's observation.

Treatment success is assessed on study day 7 to 14 after treatment. Ananimal is classified as successfully treated treatment if it has arectal temperature of less than 39.5° C., bacteriological negative andnon fetid vulval discharge.

Example 9 Pharmacokinetics of Bovine Casein Hydrolysate

In Vivo Bacterial Activity, Transmigration and Phagocytes Activity

A preliminary measurement of dose-response on bacterial activity,transmigration and phagocytes activity is performed after intrauterineinfusion of bovine casein hydrolysate to dairy cows with confirmeduterine infections (clinical, bacteriological examination of uterineswabs) compared to controls.

Dose-depended response is assessed in three study or case groups, eachgroup consisting of three cows. Each group receives the followingintrauterine dose of bovine casein hydrolysate about 24 days postpartum: 0.5 mg/kg (Group I), 2.5 mg/kg (Group II) and 4.4 mg/kg (GroupIII). The control group (Group IIII) consists of cows receiving at thesame time as the study groups a solution as described in the protocol byintrauterine administration.

Endometrial cytology samples for determination of endometrialpolymorphonuclear neutrophil granulocytes and phagocytic activity arecollected AT 0, and around 12 and 24 hours after dosing. Furtherfollow-up determinations are performed from endometrial cytology samplesat 2, 5 and 7 days.

Cells are harvested from uterine lumen and counted using fluorescencemicroscopy. For the assessment of cell viability, the total number ofviable polymorphonuclear neutrophil granulocytes after co-culture withbacterial products are determined with a quantitative flow cytometricstandard cell dilution assay applying propidium iodide for theassessment of cell viability according the method by Hendrick A et al.(Hendricks A., Leibold W., Kaever V., Schuberth H.-J. Prostaglandin E2is variably induced by bacterial superantigens in bovine mononuclearcells and has a regulatory role for the T cell proliferative response.Immunobiology, 201:493-505 (2000)). For cultured bacteria, thesupernatants of for example, Escherichia coli and Arcanobacteriumpyogenes, after centrifugation 15,000 g, 20 minutes, 4° C., is removed,sterile-filtered (0.5 μm), and stored in aliquots at −80° C. Sedimentbacterial are washed twice in sterile phosphate-buffered saline (PBS)and are disrupted into fragments by a French Pressure Cells and Press.Complete killing of the bacteria is judged by cultivate in blood agar(24 hours, 37° C., 5% CO₂ in air). The concentration of supernatants andbacterial fragments are expressed in equivalents of colony forming units(CFU) present in the suspension before preparation. For example,Escherichia coli preparation containing 3 μg/ml of lipopolysaccharide(LPS) (equivalent to 2×10⁸ CFU/ml). Lipopolysaccharide contained inArcanobacterium pyogenes are below the detection limits (<0.001 μg/ml).

In Vitro Chemotaxis

Chemotaxis of polymorphonuclear neutrophil granulocytes is also studied(in triplicates) in an acrylic transmigration chamber using 3 mm porepolycarbonate filters. The lower well of each of the transmigrationchambers is filled with about 30 mL of bovine casein hydrolysate atdifferent peptide concentrations, and with 300 μL of the chemokinerhIL-8 at concentrations of 0, 10, 25, 50, 100, 150, 200 rhIL-8 ng/mLmedium and subsequently each well is under layered with 115 mL undilutedisotonic Percoll (100%). At the end of the assay, residual non-migratedpolymorphonuclear neutrophil granulocytes cells from each upper well arecollected. Upon removal of the membrane, migrated cells are harvestedfrom the lower well of each chamber and cells from lower well and upperwells are quantified by flow cytometry using a Standard Cell

Dilution Assay (SCDA) which quantify any subset of phenotypicallydefinable, viable cells in heterogeneous populations using a FACScanflow cytometer (Pechhold K., Pohl T., Kabelitz D. Rapid quantificationof lymphocyte subsets in heterogeneous cell populations by flowcytometry. Cytometry, 16:152-159 (1994)).

For subsequent in vitro experiments with migrated polymorphonuclearneutrophil granulocytes (evaluation of functional and phenotypicalproperties) the harvested cells are washed once with phosphate buffersaline (100×g, 4° C., 8 min) and re-suspended in cell culture medium(rhIL-8 ng/ml medium and undiluted isotonic Percoll (100%)) at 2×10⁶cells/ml where polymorphonuclear neutrophil will have >95% purity, >95%viability).

In Vitro Phagocytic Activity

A commercially available nonviable Streptococcus zooepidemicussuspension is labeled with fluoresceinisothiocyanat for phagocyticactivity. The polymorphonuclear neutrophil granulocytes with ingestedfluoresceinisothiocyanat-labeled streptococci is distinguished fromnon-phagocytic polymorphonuclear neutrophil granulocytes by increasedgreen fluorescence after flow cytometric acquisition and analysis.

Example 10 In vitro Model of Generation of Reactive Oxygen Species

Polymorphonuclear neutrophil granulocytes obtained from uterine scrapingare stimulated with phorbol myristate acetate (PMA, 300 nmoL/1). The PMAserves as a receptor-independent activator of proteinkinase C and, thus,as a stimulator of reactive oxygen species (ROS)-formation. Phosphatebuffer saline (PBS) instead of PMA serves as negative control. After 15min (37° C. 5% CO₂ in air) of PMA induced activation of the leukocytes,the cells are loaded with the nonfluorescent dye dihydrorhodamin 123(DIM 123, 15 μg/mL) is added to each of the wells. Short agitation and athird incubation phase (37° C. 5% CO₂ in air) of 15 min, allow foroxidative transformation of DHR 123 depending on the amount of ROSgenerated, into the green fluorescent rhodamin 123 via oxidationcatalyzed by cellular myeloperoxidase. Subsequently all samples areplaced on ice and kept in the dark until analysis by flow cytometry(2000 PMN) by determinate of the relative (mean) fluorescence intensity(Emmendörffer A., Hecht M., Lohmann-Matthes M. L., Roesler J. A fast andeasy method to determine the production of reactive oxygen intermediatesby human and murine phagocytes using dihydrorhodamin 123. J. Immunol.Method 131:269-275 (1990)).

The invention claimed is:
 1. A method for treatment of an uterineinfection, the method comprising transcervical intrauterineadministration to a female livestock mammalian animal in need oftreatment of uterine infection, an amount of at least one caseinpeptide, the amount being effective to treat an uterine infection in themammalian animal, wherein said administration of the at least one caseinpeptide is in combination with a pharmaceutical carrier providing saidtranscervical intrauterine administration of the at least one caseinpeptide.
 2. The method of claim 1, wherein the at least one caseinpeptide comprises one or more peptide fragments of β-casein, αS1-casein,αS2-casein, κ-casein.
 3. The method of claim 1, wherein the at least onecasein peptide is a casein hydrolysate.
 4. The method of claim 1,wherein at least one casein peptide comprises a phosphopeptide.
 5. Themethod of claim 1, wherein the at least one casein peptide comprises anamino acid sequence selected from the group consisting of SEQ ID NO:2,SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6.
 6. The method ofclaim 1, wherein the uterine infection is selected from the groupconsisting of toxic puerperal metritis, clinical metritis, perimetritis,clinical endometritis, sub-clinical endometritis, pyometra, cervicitis,vaginitis and vulvitis.
 7. The method of claim 6, wherein the uterineinfection is selected from the group consisting of toxic puerperalmetritis, clinical metritis, clinical endometritis and sub-clinicalendometritis.
 8. The method of claim 1, wherein the uterine infection isa post-partum infection.
 9. The method of claim 1, wherein the femalemammalian animal is a lactating animal.
 10. The method of claim 9,wherein the female mammalian animal is selected from the groupconsisting of nulliparous, primiparous or multiparous lactating animal.11. The method of claim 1, wherein the female mammalian animal is a cow.12. The method of claim 1, wherein the administration is by injection orinfusion.
 13. The method of claims 1, wherein the at least one caseinpeptide is administered at a time period between calving.
 14. The methodof claim 13, wherein the time period comprises up to 120 days followingcalving.
 15. The method of claim 14, wherein the time period comprisesup to 70 days following calving.
 16. The method of claim 14, wherein thetime period is from day up to 33 days following calving.
 17. The methodof claim 1, wherein the administration comprises a single doseadministration.
 18. The method of claim 1, wherein the administrationcomprises two or more administrations of the at least one caseinpeptide, the two or more administrations comprise a time interval offrom 1 hour to 14 days.
 19. The method of claim 18, wherein theadministration comprises from two to six administrations.
 20. The methodof claim 1, wherein the amount of said at least one casein peptideeffective to treat said uterine infection is between 10 ng/ml to 500mg/ml.